Self-Albumin Camouflage of Carrier Protein Prevents Nontarget Antibody Production for Enhanced LDL-C Immunotherapy.

Elevated low-density lipoprotein cholesterol (LDL-C) increases the risk of atherosclerotic cardiovascular disease. Peptide-based PCSK9 vaccines have shown a promising prospect of reducing LDL-C. In peptide vaccine (pVax) design, the peptide antigens need to conjugate with carrier protein (CP). However, CP incorporation can induce undesirable anti-CP antibodies, which sterically mask peptide epitopes from being recognized by specific B cells and impair subsequent therapeutically antibody production. This epitopic suppression has posed a barrier in clinical translation of conjugate vaccines all along. A model CP (keyhole limpet hemocyanin, KLH) is herein camouflaged with serum albumin (SA) into hybrid nanocarriers (SA@N), with PCSK9 peptide being anchored onto the surface to form nanovaccine (SA@NVax). Such camouflage of KLH via high "self" SA coverage is able to inhibit KLH from extracellular immune recognition and prevent detectable anti-KLH antibody production. Furthermore, the nanovaccine around 70 nm stabilized by intermolecular disulfide network is ideal for internalization and biodegradation by antigen presenting cells as well as better retention in draining lymph nodes and spleen. As expected, the SA@NVax efficiently primes higher anti-PCSK9 IgG antibody titer than PCSK9 pVax.

Carrier priming with CRM 197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: biophysical and immunochemical interpretation.

Glycoconjugate vaccines play an enormous role in preventing infectious diseases. The main carrier proteins used in commercial conjugate vaccines are the non-toxic mutant of diphtheria toxin (CRM197), diphtheria toxoid (DT) and tetanus toxoid (TT). Modern childhood routine vaccination schedules include the administration of several vaccines simultaneously or in close sequence, increasing the concern that the repeated exposure to conjugates based on these carrier proteins might interfere with the anti-polysaccharide response. Extending previous observations we show here that priming mice with CRM197 or DT does not suppress the response to the carbohydrate moiety of CRM197 meningococcal serogroup A (MenA) conjugates, while priming with DT can suppress the response to DT-MenA conjugates. To explain these findings we made use of biophysical and immunochemical techniques applied mainly to MenA conjugates. Differential scanning calorimetry and circular dichroism data revealed that the CRM197 structure was altered by the chemical conjugation, while DT and the formaldehyde-treated form of CRM197 were less impacted, depending on the degree of glycosylation. Investigating the binding and avidity properties of IgGs induced in mice by non-conjugated carriers, we found that CRM197 induced low levels of anti-carrier antibodies, with decreased avidity for its MenA conjugates and poor binding to DT and respective MenA conjugates. In contrast, DT induced high antibody titers able to bind with comparable avidity both the protein and its conjugates but showing very low avidity for CRM197 and related conjugates. The low intrinsic immunogenicity of CRM197 as compared to DT, the structural modifications induced by glycoconjugation and detoxification processes, resulting in conformational changes in CRM197 and DT epitopes with consequent alteration of the antibody recognition and avidity, might explain the different behavior of CRM197 and DT in a carrier priming context.